The protein Cu/Zn superoxide dismutase (SOD1) isknown to function as a dimer, but its concentration in cells (∼50μM) and the dimerization constant (K_(d) of 500 μM) results suggestthat it exists in a monomer-dimer ...The protein Cu/Zn superoxide dismutase (SOD1) isknown to function as a dimer, but its concentration in cells (∼50μM) and the dimerization constant (K_(d) of 500 μM) results suggestthat it exists in a monomer-dimer equilibrium. It is unclear how theoligomeric state of SOD1 changes when cells are initially exposed tohigh levels of extracellular oxidative stress. To address this problem,we introduced the single-molecule ffuorescence anisotropy (smFA)assay to explore SOD1 oligomeric states in live COS7 cells. smFAspeciffcally probes the ffuorescence polarization changes caused bymolecular rotations where the fast-rotating molecules (either due tosmaller hydrodynamic volume or less viscous environments)deteriorate the emission polarization and thus lower the anisotropy. After validating that smFA is effective in distinguishingmonomeric and dimeric ffuorescence proteins, we overexpressed SOD1 in live COS7 cells and investigated how its oligomeric statechanges under basal, 2 h, and 24 h 100 μM H_(2)O_(2) treatments. We found that treating cells with H_(2)O_(2) promotes SOD1 dimerizationand decreases cellular viscosity in 2 h. Interestingly, prolonged H_(2)O_(2) treatments show similar results as the basal conditions,indicating that cells return to a steady state similar to the basal state after 24 h, despite the presence of H_(2)O_(2). Our resultsdemonstrate that SOD1 changes its oligomeric state equilibrium in response to extracellular oxidative stresses. smFA will open newopportunities to explore the relationship between the SOD1 oligomer state and its H_(2)O_(2)-based signaling and transcription regulationroles.展开更多
目的探讨山奈酚(kaempferol,KAE)对慢性脑缺血大鼠的作用及其机制。方法采用双侧颈总动脉永久性结扎(bilateral common carotid arteries occlusion,2VO)建立大鼠慢性脑缺血动物模型;Morris水迷宫、抓握实验检测大鼠行为学;尼氏染色及H...目的探讨山奈酚(kaempferol,KAE)对慢性脑缺血大鼠的作用及其机制。方法采用双侧颈总动脉永久性结扎(bilateral common carotid arteries occlusion,2VO)建立大鼠慢性脑缺血动物模型;Morris水迷宫、抓握实验检测大鼠行为学;尼氏染色及HE染色观察脑组织病理改变;比色法检测MDA含量和SOD活性;Western blot检测脑组织DJ-1蛋白表达水平。结果与2-VO模型组比较,KAE明显改善慢性脑缺血诱导的学习记忆障碍和抓握能力损伤。此外,KAE明显减轻2VO大鼠脑组织病理损伤,增高脑组织中SOD活性,提高海马和皮层中抗氧化蛋白DJ-1的表达水平。结论KAE明显改善慢性脑缺血诱导的大鼠认知功能障碍、四肢平衡能力障碍及病理损伤,其机制可能与提高体内抗氧化系统有关。展开更多
基金supported by the National Institutes of Health(Grant No.R35GM133505)and the University of Houston.
文摘The protein Cu/Zn superoxide dismutase (SOD1) isknown to function as a dimer, but its concentration in cells (∼50μM) and the dimerization constant (K_(d) of 500 μM) results suggestthat it exists in a monomer-dimer equilibrium. It is unclear how theoligomeric state of SOD1 changes when cells are initially exposed tohigh levels of extracellular oxidative stress. To address this problem,we introduced the single-molecule ffuorescence anisotropy (smFA)assay to explore SOD1 oligomeric states in live COS7 cells. smFAspeciffcally probes the ffuorescence polarization changes caused bymolecular rotations where the fast-rotating molecules (either due tosmaller hydrodynamic volume or less viscous environments)deteriorate the emission polarization and thus lower the anisotropy. After validating that smFA is effective in distinguishingmonomeric and dimeric ffuorescence proteins, we overexpressed SOD1 in live COS7 cells and investigated how its oligomeric statechanges under basal, 2 h, and 24 h 100 μM H_(2)O_(2) treatments. We found that treating cells with H_(2)O_(2) promotes SOD1 dimerizationand decreases cellular viscosity in 2 h. Interestingly, prolonged H_(2)O_(2) treatments show similar results as the basal conditions,indicating that cells return to a steady state similar to the basal state after 24 h, despite the presence of H_(2)O_(2). Our resultsdemonstrate that SOD1 changes its oligomeric state equilibrium in response to extracellular oxidative stresses. smFA will open newopportunities to explore the relationship between the SOD1 oligomer state and its H_(2)O_(2)-based signaling and transcription regulationroles.
